Hybrid selection for sequencing pathogen genomes from clinical samples

Abstract

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla.

Sequence-based association and selection scans identify drug resistance loci in the Plasmodium falciparum malaria parasite

“…This process produces 225,623 segregating SNPs, of which 25,757 met our call rate and minor allele frequency criteria for further study (see Methods). Sequence-based SNP calling in P. falciparum is technically challenging because of its extremely AT-rich genome (12,13). In light of this finding, we validated our sequence-based approach against array-based methods by using a previously described SNP array (6) to genotype 24 of the 45 isolates.…”

“…In P. falciparum, the lack of tagging ability because of the near absence of long-range LD limits the utility of arrays for association studies. Furthermore, the small genome size of P. falciparum brings the cost of whole-genome sequencing to approximate parity with traditional genotyping arrays, and recent advances in pathogen-specific DNA-enrichment and host-specific DNA-depletion techniques for clinical samples makes the sequence-based GWAS approach more accessible and cost-effective than ever before (13,25).…”

Efficient Depletion of Host DNA Contamination in Malaria Clinical Sequencing

“…The captured pathogen DNA is pulled down and sequenced, while the nonhybridizing host contaminants are washed away. Although these techniques improve sample quality (22)(23)(24), they have numerous limitations and drawbacks. First, the oligonucleotide baits (probes) are strain specific and expensive and only partially remove the contaminating genome.…”

“…Second, optimal hybridization conditions for complete genome pulldown that avoid nonspecific capture and bias are difficult to achieve. Third, the current techniques require specialized skills and relatively large quantities (at least 2 g) of starting DNA material to complete the purification process (23). A traditional method for separating human and P. falciparum DNA involves the use of Hoechst dye and ultracentrifugation in a CsCl gradient (25).…”

Malaria Genomics in the Era of Eradication

“…Two strategies have been developed to address this challenge: processes that enrich the parasite DNA either at the time of collection or from the already extracted nucleic acid material and use of genotyping tools that do not require removal of host DNA. Approaches, including hybrid selection (Melnikov et al 2011;Bright et al 2012) and selective whole genome amplification (Leichty and Brisson 2014), enable efficient sequencing by enriching for parasite over host genetic material. Filtration methods at the time of collection have also been employed to reduce host material, with variable success (Venkatesan et al 2012).…”